The measurement of physicochemical properties, such as permeability, in a high-throughput screening environment plays an important role in the selection of the most promising biologically active molecules for lead optimization in pharmaceutical and biotechnological research and development, and in identifying active compounds with suitable distribution properties in agrochemical research and development.
In pharmaceutical research, the search for new chemical entities potentially useful as drugs takes place in three stages: exploration, discovery, and development. In the first stage, a therapeutic target is selected, and a biological screening assay developed. In the course of a year at a large pharmaceutical company, it is not uncommon to have 100,000 to 1,000,000 library compounds tested against a particular target. Of the molecules tested for biological activity, about 3000 to 10,000 are found to be active (hits). The initial part of the discovery step is called “lead” generation, where the most promising subset of the hits is selected for further testing. The selection of leads takes into account bio-pharmaceutic properties of the hits, such as measured aqueous solubility, octanol-water partition coefficients, plasma stability, human serum protein binding, cytochrome P450 inhibition (oxidative metabolism), liver microsome assay (general metabolism), and membrane permeability. These various tests filter out many molecules with unfavorable bio-pharmaceutic ADME properties (absorption, distribution, metabolism, and excretion).
ADME is the single largest cause of attrition in drug development. Methods, which can lower this high attrition rate, would benefit the industry by reducing failure rates; the pharmaceutical companies by reducing costs; and consumers by helping to get better drugs to market, in less time.
One of the common problems observed with new entities is the ability to overcome biological barriers, e.g. entering the blood circulation from the gastro-intestine or to enter the interior of a cell. This ability is routinely assessed, because it is an important pre-requisite for any substance to be transported to the site of possible pharmacological action. Decisions about the ability to develop new molecular entities are made based on such experiments. Moreover, the FDA (US Food and Drug Administration) has acknowledged the biopharmaceutical classification system [1], which has made it possible to reduce the extent of formulation work in the drug development phase, which is necessary for the registration of new drug preparations. The decision is based on solubility and permeability experiments carried out with the drug substance (“in vitro biowaiver” [2]).
Therefore, drug permeability screening has become a routine method for the pharmaceutical industry. Many big pharmaceutical companies do these experiments in-house, while others may use one of the numerous specialized contract service labs for such studies.
Standard cell models for drug permeability studies are based on a microwell plate format where cell lines are grown on filter insert supports. The most common cell model of this type is called Caco-2 assay [3], which is frequently used to predict human absorption of the drugs from the intestine. The cells are grown to make a tight barrier of a single layer of cells, a process, which takes approx. 3 weeks during which the cells need frequent attendance in a specialized cell culture lab. For the permeation test, these cell layers on their supports are exposed to the drug solution and the flux of the drug molecules through the cell layer is measured. The assay includes both the passive and the active transport. Numerous specialized labs provide contract service for such studies, if the facilities are not available in-house. The results of such permeation experiments frequently show large variation. An estimation of the very best reproducibility level that can be expected is approx. 50%, as can be estimated from values published by a contractor lab cyprotex [4]. Commonly much more variability is observed. Unfortunately, in addition, the comparability between labs tends to be very poor.
The Franz Cell apparatus [5] is also widely used and the devices needed for the tests are commercially available in different designs. The cell is based on diffusion chambers made of two borosilicate glass component cells.
For the experiments, typically biological tissue is extracted from animals, either kept alive (including active transport) or not (passive transport only), and placed in between the 2 compartments. Flux of drug molecules is measured by collecting samples from the acceptor compartment over time. The handling of this model is laborious and may be unpleasant, and the results are biased by biological variation.
Non-cell based models are the PAMPA (parallel artificial membrane permeability assay) as well as the PVPA (phospholipid vesicle-based permeation assay) model. They are both based on multiwell plates format. The PAMPA model is frequently used for drug screening. It is exclusively commercially available in several variations, with secret composition of both the barrier and the solvents used. In order to make the model robust enough for high throughput, a porous substrate is impregnated with a lipophilic phase to mimic cell membranes. In general, the well plates comprise filter inserts soaked in organic solution of lipids in dodecane, or hexadecane as a lipid compound, or more complex mixtures such as the special tri-layer structures (lipid/oil/lipid). The different PAMPA set-ups and methods of the model vary according to the aim of the study. For the experiment, the membranes need to be freshly soaked with the provided solutions.
The PVPA model (phospholipid vesicle permeation assay) is also a lipid-based model. However, it contains liposomes (closed vesicles of phospholipid bilayers) which resemble cells, in a tight packing. It is believed that this set-up is much more comparable to physiologic properties, and it has shown to predict the passive transport in human oral absorption better than the PAMPA model and as good as the CaCo2-cell assays (passive transport only), with a good reproducibility (about 10% st.dev. for n=3, maximum value 25% [6]).
The method can be automatized for medium throughput; however, the barriers need to be prepared by a laborious procedure, which is not trivial. In brief, liposome dispersions of certain sizes are prepared by extrusion, and deposited on a filter support by centrifugation. The liposomes are added in consecutive steps, starting with the smallest ones. Freeze-thaw cycles are used to tighten the barriers by liposome fusion. The exact procedure is published [6]. The model is used by several research groups, however, it is not commonly used in industry. It has recently been refined to use other lipid compositions and solvents to mimic other biological barriers, e.g. the skin. [7]
PAMPA is commercially available in several variants as complete kits, with long storage time, and it is fast to use. However, the PAMPA barriers are less able to resemble in vivo permeation properties than rather a partitioning into non-physiological oil phases. The barriers show limited resistance to some excipients. The devices only come in the microtiterplate format. The kits are very expensive. PVPA is from a mechanistic point of view based on similar principles of the permeation layer being composed of physiological lipids. However, the experiments are rather laborious to carry out. This is connected to the tedious preparation procedure of the barriers and their lack of long-term stability (they can be frozen and should be used within 2 weeks). The barriers are not commercially available. During an experiment, the barriers cannot be used over a long time due to mechanical reasons, and they are incompatible with many excipients.
The Frantz cell model using tissues needs to standardize the tissues (fresh or frozen). Hence, there is an intrinsic variability according to the origin of the tissue samples. Accessibility of tissues is another serious restriction. Cell-based systems need to be grown in special lab facilities over long time periods (which is a matter of weeks). They include the possibility to study active transport, however, the passive transport is always present and needs to be studied separately. Cell based assays lack reproducibility; the inter-lab variance is so high that data should not be compared even if the same cell line is used. Due to the sensitivity of the cells, they cannot be used with concentrated formulations and some excipients.
Corti et al [8] describe the development of an assay for predicting permeability of drugs across artificial membranes, which is similar to the PAMPA approach. The device used for the assay is a Sartorius absorption simulator model comprising i) a donor compartment for adding a composition comprising the compound, ii) a barrier comprising a porous support phospholipid impregnated with a mixture of phospholipids (<2%), cholesterol and mainly the solvent octanol (96%) on said support, and iii) an acceptor compartment for accepting the compound after permeation of the barrier.
EP1779921A1 discloses an improved phospholipid membrane for use in the PAMPA assay. A porous support made from, e.g. polycarbonate, is initially coated with a non-volatile support liquid, e.g. hexadecane. A phospholipid solution in a volatile solvent is applied to the surface of the porous support and the solvent is allowed to evaporate, leaving a lipid layer on the porous support. The amount of support liquid is preferably minimized (section [0020]) and depending on the amount used, the phospholipid layer may form above the porous support, on the porous support, or within the pores of the support. EP1779921A1 further describes the use of the phospholipids in the PAMPA assay, which is described as a multiplex setup with donor compartments separated from acceptor compartments by way of a barrier comprising the porous support with a phospholipid layer adhering to said support.
The above-mentioned problems have been solved by the present invention by a novel biomimetic artificial barrier for drug permeability studies. The barrier is cost effective and easy to use, and has a much higher chemical resistance in comparison to all other available models. The barriers of the present invention can be adapted to Franz cell diffusion chambers and permeability coefficients of drugs can be measured. Moreover, tightness of the barrier in respect to hydrophilic markers, the resistance of the barriers to proton permeation (pH changes) and shelf-life stability in terms of functionality render them very applicable.
The present invention also seeks to solve the problems associated with prior art barriers when e.g. surfactants and co-solvents are present. Thus it is an objective of the present invention to provide a barrier that can withstand surfactants and co-solvents without affecting the integrity of the barrier and thereby its permeability.